JPWO2018199080A1 - Imaging flow cytometer - Google Patents

Abstract

The imaging flow cytometer includes at least one flow path through which an observation target flows, a light source that irradiates the flow path with a band-like excitation light, and fluorescence from the observation target that has passed through the position where the excitation light is irradiated. A three-dimensional image generating unit that generates a three-dimensional image of the observation target based on a plurality of captured images of the cross-section captured by the imaging unit. An image generation unit.

Description

The present invention relates to an imaging flow cytometer.
This application claims priority based on Japanese Patent Application No. 2017-090798 for which it applied to Japan on April 28, 2017, and uses the content here.

2. Description of the Related Art Conventionally, there are known a flow cytometry method in which an observation target is fluorescently stained and the characteristics of the observation target are evaluated based on a total amount of the fluorescence luminance, and a flow cytometer using the flow cytometry method (for example, Patent Reference 1). In addition, a fluorescence microscope and an imaging cytometer that evaluate fine particles of cells, bacteria, and the like as observation objects by using images are known.
In addition, there is known an imaging flow cytometer that captures morphological information of fine particles at a high speed at the same throughput as a flow cytometer (for example, Patent Document 2). In addition, a polygonal optical microscope that irradiates a band-shaped excitation light to a cell serving as an observation target to obtain an image of a plane at a different angle with respect to an irradiation surface irradiated with the excitation light or a three-dimensional image of the observation target. Are known (for example, Patent Documents 3 and 4).

Japanese Patent No. 5534214 U.S. Pat. No. 6,249,341 US Patent Application Publication No. 2015/0192767 U.S. Pat. No. 5,852,203

In a conventional imaging flow cytometer, a two-dimensional image of a cell is generated by imaging a surface irradiated with excitation light. However, a method of generating a three-dimensional image of a cell under a condition of observing the cell at a high speed such as an imaging flow cytometer has not been known.
An object of the present invention is to provide an imaging flow cytometer that generates a three-dimensional image of an observation target at high speed.

  One embodiment of the present invention provides at least one flow path through which an observation target flows, a light source that irradiates the flow path with a band-like excitation light, and the observation target that has passed a position irradiated with the excitation light. An imaging unit that captures an image of a cross section of the observation target by imaging fluorescence from the object; and a three-dimensional image of the observation target based on a plurality of captured images of the cross section captured by the imaging unit. The imaging flow cytometer includes a three-dimensional image generation unit that generates a three-dimensional image.

  In one embodiment of the present invention, in the imaging flow cytometer, the observation target is sorted based on information indicating a form of the observation target indicated by the cross section captured by the imaging unit.

  In one embodiment of the present invention, in the above-described imaging flow cytometer, the flow path is a plurality of flow paths arranged in parallel, and the excitation light is applied to the plurality of flow paths. The imaging unit images the cross section of the observation target flowing through each of the plurality of flow paths.

  Further, one embodiment of the present invention is the above-described imaging flow cytometer, wherein the light modulation includes a plurality of regions having different optical characteristics on an optical path between the light source and the image sensor for detecting the intensity of the fluorescence. A section, and the imaging section re-creates the image of the cross section of the observation target as a captured image based on the intensity of the fluorescence detected by the imaging element and the optical characteristics of the light modulation section. Constitute.

  According to the present invention, it is possible to provide an imaging flow cytometer that generates a three-dimensional image of an observation target at high speed.

It is a figure showing the appearance composition of a cell measurement system. FIG. 2 is a diagram illustrating an example of a functional configuration of the cell measurement system according to the first embodiment. It is a figure showing the section of the cell which an image sensor picks up. It is a figure which shows the order which a three-dimensional image production part combines a cross-sectional image. 5 is a flowchart showing an example of the operation of the imaging flow cytometer. It is an example of a plurality of flow paths. It is a figure showing an example of functional composition of a cell measurement system of a 2nd embodiment. 5 is a flowchart showing an example of the operation of the imaging flow cytometer. It is a figure which shows an example of the structure which looked at the flow path which sorts a cell from + z-axis direction.

<First embodiment>
Hereinafter, a first embodiment of a cell measurement system will be described with reference to the drawings.
FIG. 1 is a diagram illustrating an external configuration of the cell measurement system 1.

  The cell measurement system 1 includes an imaging flow cytometer 20 and a display unit 10. The imaging flow cytometer 20 includes at least one flow path through which the observation target flows. In the present embodiment, a case will be described where the observation target is a cell, but the observation target is not limited to a cell. The observation target may be any object that transmits light. The cells are cells that have been fluorescently stained.

  The imaging flow cytometer 20 generates a three-dimensional image of the cells flowing through this flow channel. The display unit 10 displays a three-dimensional image generated by the imaging flow cytometer 20. The display unit 10 includes, for example, a liquid crystal display, and displays various images. The image displayed on the display unit 10 includes a three-dimensional image of cells generated by the imaging flow cytometer 20.

<Functional configuration of imaging flow cytometer 20>
Next, a functional configuration of the imaging flow cytometer 20 will be described with reference to FIG.
FIG. 2 is a diagram illustrating an example of a functional configuration of the cell measurement system 1 according to the first embodiment.

  FIG. 2 shows an xyz coordinate system as a three-dimensional orthogonal coordinate system. In the present embodiment, the x-axis direction is a depth direction of the flow path 21. The y-axis direction is the direction in which the cells CL in the flow channel 21 flow. The cell CL is caused to flow in the + y direction along the y-axis. The z-axis direction is a direction orthogonal to the flow path 21 and a height direction of the flow path 21.

  The imaging flow cytometer 20 includes an imaging unit 22, a storage unit 24, and a three-dimensional image generation unit 25, in addition to the above-described channel 21.

The imaging unit 22 includes a light source 26, an objective lens OG, an imaging element 27, an optical element L1, an optical element L2, an optical element L3, and a control unit 200.
The light source 26 irradiates the band-shaped excitation light LS to the channel 21. Specifically, the light source 26 irradiates the channel 21 with coherent light having a band shape. The band-like excitation light LS is a band-like excitation light by narrowing the coherent light. The band-shaped excitation light is coherent light having a width longer than the diameter of the sample in the x-axis direction and adjusted to a thickness shorter than the diameter of the sample in the y-axis direction. In the present embodiment, the light source 26 irradiates the band-shaped excitation light LS to the channel 21 via the dichroic mirror M1 and the objective lens OG.
In the following description, the position of the flow path 21 where the excitation light LS is irradiated is also described as the irradiation position. The cells CL that have passed this irradiation position emit light when the fluorescent molecules are excited by the excitation light LS. The light due to this emission is the fluorescence FL.
The imaging unit 22 captures an image of the fluorescence FL from the cell CL that has passed through the position irradiated with the excitation light LS. Thereby, the imaging unit 22 captures an image of a section of the cell CL. Here, a certain cross section is a plane where the fluorescent molecules are excited by the excitation light LS. In other words, a certain cross section is a surface in a direction different from the direction in which the sample flows through the flow channel 21. The configuration of the optical system for imaging a certain cross section of the cell is, for example, the configuration disclosed in U.S. Patent Application Publication No. 2015/0192767 and U.S. Patent No. 5,852,203.

  The objective lens OG collects the fluorescent light FL from the cell CL. The objective lens OG is an objective lens arranged at a position where the irradiation position is focused. In the following description, a position where the irradiation position is focused on is also referred to as a focal position FP. The objective lens may be a dry objective lens or an immersion objective lens. The immersion objective lens is an oil immersion lens, a water immersion lens, or the like.

  The fluorescence condensed by the objective lens OG forms an image on the image sensor 27 via the optical element L1, the optical element L2, and the optical element L3. In this example, the fluorescence collected by the objective lens OG is formed as an image VRI via the optical element L1. This image VRI is an image obtained by inverting the cell CL in the x-axis direction and the z-direction. The imaging element 27 captures the image VRI via the optical element L2 and the optical element L3.

  The image formed on the image sensor 27 is a surface in a direction different from the direction of the surface on which the excitation light LS is irradiated on the flow path 21. In the present embodiment, a case where the image formed on the image sensor 27 is a surface orthogonal to the irradiation surface of the excitation light LS will be described. This surface is a cross section of the cell CL.

<About the cross section>
Here, an example of a cross section of the cell CL imaged by the imaging element 27 will be described with reference to FIG.
FIG. 3 is a diagram illustrating a cross section of the cell CL imaged by the image sensor 27.
FIG. 3A is a diagram illustrating the relationship between the position of the excitation light LS and the position of the cell CL. The excitation light LS emitted from the light source 26 is emitted in the z-axis direction. That is, the excitation light LS is emitted in the direction of the surface AP.
FIG. 3B is a diagram of the cell CL viewed from FIG. 3A viewed from the y-axis direction. The excitation light LS spreads in a band shape in the y-axis direction and the x-axis direction. The cells CL pass through the flow path 21 and pass through the excitation light LS spread in a band shape. Here, the width W1 of the excitation light LS shown in FIG. 3A is smaller than the width W2 of the excitation light LS shown in FIG. Specifically, the width W1 is 2 to 3 μm.
FIG. 3C is a diagram illustrating a cross section of the cell CL imaged by the image sensor 27. The imaging element 27 captures an image of a plane LP that is a plane orthogonal to the plane AP.

Returning to FIG. 2, the image sensor 27 images the cross section of the cell. Here, the image sensor 27 is specifically a line scan camera. The line scan camera acquires the detected light intensity for each pixel row of the image sensor 27 in the vertical or horizontal direction. By using a line scan type image sensor, the image sensor 27 can acquire only the light intensity of a pixel row in a region where an image is formed. Accordingly, the imaging device 27 can reduce the time for acquiring the light intensity of the pixel row in the region where no image is formed, as compared with an imaging device other than the line scan type. Further, it is possible to reduce the time for image processing for generating a captured image based on the light intensity acquired from the image sensor 27. Thereby, the image sensor 27 can acquire an image at high speed.
In this example, the image sensor 27 is an image sensor configured from sCMOS (Scientific CMOS; CMOS for scientific measurement) or the like. The sCMOS can capture images at a higher speed and with higher image quality than an image sensor constituted by a conventional CCD or CMOS.
The imaging element 27 supplies the captured image to the image acquisition unit 23.

The control unit 200 includes, for example, a CPU and a GPU (Graphics Processing).
Unit), an FPGA (field-programmable gate array), and the like, and perform various calculations and exchange of information. The control unit 200 includes the image acquisition unit 23 as a functional unit.
The image acquisition unit 23 acquires a captured image from the image sensor 27. The image acquisition unit 23 causes the storage unit 24 to store the captured image acquired from the image sensor 27 as a cross-sectional image PIC. The storage unit 24 stores the order in which the cross-sectional images PIC are captured.

  The three-dimensional image generation unit 25 acquires a plurality of cross-sectional images PIC from the storage unit 24. The three-dimensional image generation unit 25 generates a three-dimensional image of the cell CL based on the plurality of cross-sectional images PIC acquired from the storage unit 24. Specifically, the three-dimensional image generation unit 25 combines in the order in which the cross-sectional images PIC are captured in the −y-axis direction. This cross-sectional image PIC is an image obtained by imaging the plane LP described above.

Here, the order in which the three-dimensional image generation unit 25 combines the cross-sectional images PIC will be described in detail with reference to FIG.
FIG. 4 is a diagram illustrating the order in which the three-dimensional image generation unit 25 combines the cross-sectional images PIC.
FIG. 4A is a diagram illustrating an example of a correspondence relationship between a cross section of a cell CL and a cross-sectional image.
The cell CL moves in the + y-axis direction. The imaging unit 22 sequentially captures n cross-sectional images PIC from the cross-section CS1 to the cross-section CSn of the cell CL. N of the cross section CSn is an integer of 1 or more.
The storage unit 24 stores a section image PIC1 of the section CS1 to a section image PICn of the section CSn.

FIG. 4B is a diagram illustrating the order in which the three-dimensional image generation unit 25 combines the cross-sectional images PIC1 to PICn. The three-dimensional image generation unit 25 combines in the −y-axis direction in the order in which the cross-sectional images PIC are captured. This is because the cell CL moves in the + y-axis direction. Note that the order of the connection is changed according to the moving direction of the cell CL.
FIG. 4B shows an XYZ coordinate system as a three-dimensional orthogonal coordinate system. This XYZ coordinate system is a coordinate system of a three-dimensional image generated by the three-dimensional image generation unit 25. The three-dimensional image generation unit 25 combines the cross-sectional images PIC1 to PICn in a state where the XYZ coordinate system is associated with the xyz coordinate system. Specifically, the three-dimensional image generation unit 25 combines the X direction and the x direction with the same direction. The three-dimensional image generation unit 25 combines the Y-axis direction and the Y-axis direction in the same direction. The three-dimensional image generation unit 25 combines the Z-axis direction and the Z-axis direction in the same direction. In addition, the three-dimensional image generation unit 25 generates a three-dimensional image of the cell CL by sequentially connecting the cross-sectional images PIC1 to PICn in the −Y-axis direction.

  Returning to FIG. 2, the three-dimensional image generation unit 25 causes the display unit 10 to display the generated three-dimensional image of the cell CL. The display unit 10 displays a three-dimensional image of the cell CL.

<Overview of the operation of the imaging flow cytometer 20>
Next, an outline of an operation procedure of the imaging flow cytometer 20 will be described with reference to FIG.
FIG. 5 is a flowchart showing an example of the operation of the imaging flow cytometer 20. The operation procedure shown here is an example, and the operation procedure may be omitted or an operation procedure may be added.

(Step S10) The imaging element 27 always captures an image at the focal position FP. The image acquisition unit 23 acquires a signal from the image sensor 27. The signal is a signal indicating the above-described cross-sectional image PIC.
(Step S20) The image acquisition unit 23 determines whether there is a change in the signal acquired from the image sensor 27. Specifically, the image acquisition unit 23 stores a pre-detection signal that is a signal in a state where the cells CL are not flowing through the flow path. The image acquisition unit 23 compares the pre-detection signal with the signal acquired from the image sensor 27. Specifically, when there is no predetermined difference between the pre-detection signal and the signal obtained from the image sensor 27, the image obtaining unit 23 determines that there is no change in the signal. When there is a predetermined difference between the pre-detection signal and the signal obtained from the image sensor 27, the image obtaining unit 23 determines that there is a change in the signal.

(Step S20; NO) When determining that there is no change in the signal, the image acquisition unit 23 repeats the processing from step S10.
(Step S20; YES) When determining that there is a change in the signal, the image acquisition unit 23 causes the storage unit 24 to store the signal acquired from the image sensor 27 as a cross-sectional image PIC (Step S30).

(Step S40) The image acquisition unit 23 acquires a signal from the image sensor 27.
(Step S50) Based on the signal acquired from the image sensor 27, the image acquisition unit 23 determines whether or not the cross section of the cell CL has been imaged. Specifically, when there is a predetermined difference between the pre-detection signal and the signal obtained from the image sensor 27, the image obtaining unit 23 determines that the cross section of the cell has not been imaged. When there is no predetermined difference between the pre-detection signal and the signal acquired from the image sensor 27, the image acquisition unit 23 determines that the imaging of the cross section of the cell has been completed.

(Step S50; NO) When determining that the imaging of the cross section of the cell has not been completed, the image acquiring unit 23 repeats the processing from step S30.
(Step S50; YES) When determining that the imaging of the cross section of the cell has been completed, the image acquiring unit 23 executes the process of step S60.

(Step S60) The three-dimensional image generation unit 25 acquires a plurality of cross-sectional images PIC from the storage unit 24. The three-dimensional image generation unit 25 generates a three-dimensional image by combining a plurality of cross-sectional images PIC obtained from the storage unit 24.
(Step S70) The three-dimensional image generation unit 25 causes the display unit 10 to display the generated three-dimensional image. The imaging flow cytometer 20 ends the processing.

<Summary of First Embodiment>
As described above, the imaging flow cytometer 20 includes the flow path 21, the imaging unit 22, and the three-dimensional image generation unit 25. The imaging unit 22 captures a cross section of the cell CL flowing through the flow channel 21 as a cross section image PIC. The three-dimensional image generation unit 25 generates a three-dimensional image by combining a plurality of cross-sectional images PIC captured by the imaging unit 22. Thereby, the imaging flow cytometer 20 can generate a three-dimensional image of the cell CL.

The image sensor 27 included in the image capturing unit 22 is an image sensor configured by sCMOS. The sCMOS can capture images at a higher speed than an image sensor configured by a CMOS or a CCD. The imaging device 27 can capture a large number of images per second.
In addition, sCMOS can generate a high-quality captured image with reduced noise as compared with an image sensor configured by CMOS or CCD. Thereby, the imaging flow cytometer 20 can generate a detailed three-dimensional image of the cell CL.

  In the above description, the configuration in which the imaging unit 22 includes the objective lens OG, the optical element L1, the optical element L2, and the optical element L3 has been described, but the configuration is not limited thereto. The imaging unit 22 may have a configuration disclosed in, for example, US Patent Application Publication No. 2015/0192767.

In addition, the flow path 21 may be a plurality of flow paths arranged in parallel as shown in FIG.
FIG. 6 is an example of a plurality of flow paths.

FIG. 6A is a diagram of the plurality of flow paths viewed from the y-axis direction.
The channels from the channel 21a to the channel 21d are arranged in parallel in the x-axis direction. The x-axis direction is the depth direction of the flow path as described above.
The excitation light LS is emitted from the light source 26 to the plurality of channels 21. The objective lens OG collects the fluorescence FL of the cell CL passing through the irradiation position. The imaging unit 22 captures an image of a cross section of the cell CL flowing through each of the plurality of flow paths. The three-dimensional image generation unit 25 generates a three-dimensional image of the cells flowing from the flow channel 21a to the flow channel 21d from among the cross-sectional images PIC obtained by capturing the cross-sections of the plurality of cells CL.

  Thereby, the imaging flow cytometer 20 can generate a cross-sectional image PIC in which a cross-section of a plurality of cells CL is captured by one-time imaging. Thereby, the imaging flow cytometer 20 can increase the number of generated three-dimensional images of cells according to the number of flow paths imaged at one time. That is, the imaging flow cytometer 20 can generate a detailed three-dimensional image of the cell CL at higher speed.

FIG. 6B is a diagram illustrating an example of a cross section of a plurality of cells CL imaged on the image sensor 27. The cross section of the plurality of cells CL is imaged in the range of the width UW and the height UH of the imaging element 27. This range is a part of a range in which the image sensor 27 can capture an image.
The image acquisition unit 23 acquires a signal of a pixel included in the width UW and the height UH of the image sensor 27. In other words, the image acquisition unit 23 acquires signals of some pixels in a range where the image sensor 27 can image. Accordingly, the image acquisition unit 23 can acquire signals from the image sensor 27 at a higher speed than when acquiring all the signals of the pixels included in the image sensor 27.
In FIG. 6, the case where the flow path 21 has four flow paths has been described, but the present invention is not limited to this.

<Second embodiment>
Next, a second embodiment of the imaging flow cytometer will be described with reference to FIG. Note that the same components and operations as those in the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.
FIG. 7 is a diagram illustrating an example of a functional configuration of the cell measurement system 1a according to the second embodiment.

The cell measurement system 1a differs from the first embodiment in that a captured image of a cross section of the cell CL is generated by compression sensing. The compression sensing according to the present embodiment refers to reconstructing an image of a cross section of the cell CL based on the intensity of the signal of the fluorescence FL detected by one or a small number of pixel detection elements. A method for reconstructing an image of a cross section of the cell CL from a certain cross section of the cell is disclosed in, for example, International Publication No. WO 2016/136801.
The imaging flow cytometer 20a includes a light modulation unit 28 and an imaging unit 22a.

The light modulator 28 has a plurality of regions having different light characteristics. The light characteristics are, for example, characteristics relating to at least one of light transmittance, light intensity, light wavelength, and polarization. The optical characteristics are not limited to these. The light modulator 28 includes, for example, a spatial light modulator, a film on which a plurality of regions having different light characteristics are printed, and the like.
The light modulator 28 is arranged on an optical path between the light source 26 and the image sensor 27a. In the present embodiment, the light source 26 is disposed between the light source 26 and the dichroic mirror M1 on the optical path of the excitation light LS applied to the flow path 21 from the light source 26. The configuration of this arrangement is also described as the configuration of structured illumination. The structured illumination irradiates the channel 21 with the structured excitation light SLS structured by the light modulation unit 28. The cells CL flowing through the flow channel 21 emit the fluorescence FL by being irradiated with the structured excitation light SLS.

The imaging unit 22a includes an imaging element 27a and a control unit 200a.
The image sensor 27a detects the intensity of the fluorescent light FL. The imaging element 27a is, for example, a one-pixel detection element. In the present embodiment, the pixel is a part of the sCMOS described above. The imaging element 27a supplies light intensity information indicating the intensity of the detected fluorescence FL to the signal acquisition unit 29.

The control unit 200a includes, for example, a CPU, a GPU, an FPGA, and the like, and performs various calculations and exchanges information. The control unit 200a includes a signal acquisition unit 29 and an image generation unit 30 as its functional units.
The signal acquisition unit 29 acquires light intensity information from the image sensor 27a. The signal acquisition unit 29 stores the light intensity information acquired from the image sensor 27a in the order of the acquired time series. The signal acquisition unit 29 supplies the light intensity information arranged in time series to the image generation unit 30.

The image generating unit 30 reconstructs a cross-sectional image of the cell CL as a cross-sectional image PIC based on the intensity of the fluorescence FL detected by the imaging element 27a and the light characteristics of the light modulating unit 28. Specifically, the image generation unit 30 acquires the light intensity information arranged in time series from the signal acquisition unit 29. The image generating unit 30 reconstructs a cross-sectional image of the cell CL as a cross-sectional image PIC based on the time-series light intensity information acquired from the signal acquiring unit 29 and the optical characteristics of the light modulating unit 28. I do.
The image generation unit 30 causes the storage unit 24 to store the reconstructed cross-sectional image PIC.
The three-dimensional image generation unit 25 generates a three-dimensional image as in the first embodiment described above.

<Overview of the operation of the imaging flow cytometer 20a>
Next, an outline of the operation of the imaging flow cytometer 20a will be described with reference to FIG.
FIG. 8 is a flowchart showing an example of the operation of the imaging flow cytometer 20a.
The operation procedure shown here is an example, and the operation procedure may be omitted or an operation procedure may be added.

(Step S110) The imaging element 27a always detects the intensity of the fluorescence FL. The signal acquisition unit 29 acquires a signal indicating the intensity of the fluorescent light FL from the image sensor 27a.
(Step S120) The signal acquisition unit 29 determines whether there is a change in the signal acquired from the image sensor 27a. Specifically, the signal acquisition unit 29 stores a pre-detection signal that is a signal in a state where the cells CL are not flowing through the flow path. The signal acquisition unit 29 compares the pre-detection signal with the signal acquired from the image sensor 27a. Specifically, when there is no predetermined difference between the pre-detection signal and the signal acquired from the image sensor 27a, the signal acquisition unit 29 determines that there is no change in the signal. When there is a predetermined difference between the pre-detection signal and the signal acquired from the image sensor 27a, the signal acquisition unit 29 determines that the signal has changed.

(Step S120; NO) When determining that there is no change in the signal, the signal acquisition unit 29 repeats the processing from step S110.
(Step S120; YES) When determining that there is a change in the signal, the signal acquisition unit 29 stores the signals acquired from the image sensor 27a in chronological order (Step S130).

  (Step S140) The signal acquisition unit 29 acquires a signal from the image sensor 27a. The image sensor 27a determines whether there is a change in the signal acquired from the image sensor 27a. This determination is the same as in step S120 described above.

(Step S140; YES) When determining that there is a change in the signal, the signal acquisition unit 29 repeats the processing from step S130.
(Step S140; NO) When determining that there is no change in the signal, the signal acquisition unit 29 executes the process of step S150.

  (Step S150) The signal acquisition unit 29 supplies the light intensity information arranged in time series to the image generation unit 30. The image generation unit 30 acquires light intensity information arranged in time series from the signal acquisition unit 29. The image generating unit 30 reconstructs a cross-sectional image of the cell CL as a cross-sectional image PIC based on the time-series light intensity information acquired from the signal acquiring unit 29 and the optical characteristics of the light modulating unit 28. I do. The image generation unit 30 causes the storage unit 24 to store the reconstructed cross-sectional image PIC.

(Step S160) The three-dimensional image generation unit 25 acquires a plurality of cross-sectional images PIC from the storage unit 24. The three-dimensional image generation unit 25 generates a three-dimensional image by combining a plurality of cross-sectional images PIC obtained from the storage unit 24.
(Step S170) The three-dimensional image generation unit 25 causes the display unit 10 to display the generated three-dimensional image. The imaging flow cytometer 20a ends the processing.

<Summary of Second Embodiment>
As described above, the imaging flow cytometer 20a includes the light modulation unit 28, the signal acquisition unit 29, and the image generation unit 30. The signal acquiring unit 29 stores the light intensity information acquired from the image sensor 27a in a time-series order. The image generation unit 30 generates a cross-sectional image PIC including a cross-sectional image of the cell CL based on the light intensity information stored in the signal acquisition unit 29 and arranged in chronological order and the optical characteristics of the light modulation unit 28. Reconfigure. This reconstructed image is an image having a width in the y-axis direction. Thereby, the imaging flow cytometer 20a can reduce the number of times of acquiring a signal from the image sensor 27a as compared with the first embodiment described above. The imaging flow cytometer 20a can generate a three-dimensional image at higher speed.

  In the above description, the configuration in which the light modulator 28 modulates the pump light LS has been described. However, the configuration is not limited to this. For example, it may be arranged at a position where the fluorescence FL from the cell CL is modulated. For example, it is the position shown in the light modulation unit 28a of FIG. The position where the light modulation unit 28 is disposed is a position on the optical path of the fluorescent light FL before the image sensor 27a. The position at which the light modulating unit 28 is arranged is a structure arranged before the image sensor 27a on the optical path of the fluorescent light FL.

  In addition, as described in the first embodiment and the second embodiment, the control unit 200 and the control unit 200a include the GPU. As a result, the time required for image processing can be reduced as compared with the case where image processing is performed only by the CPU. Further, since the control unit 200 and the control unit 200a include the FPGA, the logic unit can perform signal processing. Accordingly, the control unit 200 and the control unit 200a can reduce the time required for signal processing as compared with the case where signal processing is performed by software. Further, the logics of the FPGAs included in the control unit 200 and the control unit 200a can be changed. Therefore, the control unit 200 and the control unit 200a can be changed to a logic circuit according to the type of signal processed. In other words, the imaging flow cytometer 20 and the imaging flow cytometer 20a can observe the observation target at a higher speed by changing the logic circuit configuration of the FPGA according to the observation target.

Note that the imaging flow cytometer 20 and the imaging flow cytometer 20a may sort the cells based on a cross-sectional image PIC or a three-dimensional image of the cell CL. In other words, the imaging flow cytometer 20 and the imaging flow cytometer 20a may sort the cells based on information indicating the morphology of the cells contained in the cross-sectional image PIC or the three-dimensional image. Sorting is to sort out predetermined cells from the measurement object flowing through the flow channel 21. This predetermined cell may be selected in advance by the user.
Here, the sorting will be described. For example, a predetermined cell as an observation target and an object different from the predetermined cell such as dust or other cells may flow in the flow path 21. Sorting is to select and extract predetermined cells from the observation target.
That is, the imaging flow cytometer 20 and the imaging flow cytometer 20a compare the information indicating the cell morphology preselected by the user with the information indicating the cell morphology included in the cross-sectional image PIC or the three-dimensional image. To determine whether the cells are cells to be sorted, and sort the cells.
In a conventional imaging flow cytometer, it takes a lot of time to generate a captured image of a cell. For this reason, the conventional flow cytometer cannot perform sorting based on the captured image. The above-described imaging flow cytometer 20 and imaging flow cytometer 20a can be sorted because the control unit 200 and the control unit 200a generate captured images at high speed and perform image processing. Further, since the imaging flow cytometer 20 and the imaging flow cytometer 20a include a plurality of flow paths 21, the number of sorted cells CL can be increased according to the number of the flow paths.

Here, a configuration for sorting cells will be specifically described with reference to FIG.
FIG. 9 is a diagram illustrating an example of a configuration in which a channel for sorting cells is viewed from the + z-axis direction.
In this example, the imaging flow cytometer 20 and the imaging flow cytometer 20a include a flow path 21a, a flow path 21b, a flow path 21c, and a flow path 21d. The flow path 21a, the flow path 21b, the flow path 21c, and the flow path 21d are arranged in parallel in the x-axis direction in the order described.
The cell CL1 flows through the channel 21a from the minus direction of the y-axis to the plus direction. The cell CL2 flows through the channel 21b from the minus direction of the y-axis to the plus direction. The cells CL3 flow through the channel 21c from the minus direction of the y-axis to the plus direction. The cell CL4 flows through the channel 21d from the negative direction of the y-axis toward the positive direction.

  The flow path 21a includes a sorting route 211a, a non-sorting route 212a, and a sorting unit 210a. The flow path 21b includes a sorting route 211b, a non-sorting route 212b, and a sorting unit 210b. The flow path 21c includes a sorting route 211c, a non-sorting route 212c, and a sorting unit 210c. The flow path 21d includes a sorting route 211d, a non-sorting route 212d, and a sorting unit 210d. In the following description, when the sorting route 211a, the sorting route 211b, the sorting route 211c, and the sorting route 211d are not distinguished, the sorting route 211 is also described. Further, in the following description, when the extra preparatory route 212a, the extra preparative route 212b, the extra preparative route 212c, and the extra preparative route 212d are not distinguished, they are also described as the extra preparative route 212. In the following description, the sorting unit 210a, the sorting unit 210b, the sorting unit 210c, and the sorting unit 210d are also referred to as the sorting unit 210 when not distinguished.

  The sorting route 211 is a channel that flows when the cells that flow in the channel 21 are predetermined cells. The unsorted route 212 is a flow channel that is flowed when cells flowing through the flow channel 21 are not predetermined cells. The sorting unit 210 switches the route through which the cells flow from the sorting route 211 and the non-sorting route 212.

In the following description, the operation of the flow path 21a will be described, but the operation of the flow path 21b, the flow path 21c, and the flow path 21d is the same.
The imaging flow cytometer 20 and the imaging flow cytometer 20a capture an image of the fluorescent light FL emitted from the cell CL1 by the excitation light LS using the image sensor 27. The imaging flow cytometer 20 and the imaging flow cytometer 20a determine whether the cell CL1 is a predetermined cell based on the captured fluorescence FL. The imaging flow cytometer 20 and the imaging flow cytometer 20a make a determination after the cell CL1 has passed the excitation light LS until it reaches the sorting unit 210a.
If the determination result indicates that the cell is a predetermined cell, the sorting unit 210a causes the cell CL1 to flow through the sorting route 211a. If the determination result indicates that the cell is not a predetermined cell, the sorting unit 210a causes the cell CL1 to flow to the unsorted route 212a. Thereby, the imaging flow cytometer 20 and the imaging flow cytometer 20a can sort predetermined cells.

  The embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and may be appropriately changed without departing from the spirit of the present invention. it can.

  Each of the above devices has a computer inside. The process of each process of the above-described apparatus is stored in a computer-readable recording medium in the form of a program, and the above process is performed by reading and executing the program by the computer. Here, the computer-readable recording medium refers to a magnetic disk, a magneto-optical disk, a CD-ROM, a DVD-ROM, a semiconductor memory, or the like. Alternatively, the computer program may be distributed to a computer via a communication line, and the computer that has received the distribution may execute the program.

Further, the program may be for realizing a part of the functions described above.
Furthermore, what can implement | achieve the function mentioned above in combination with the program already recorded on the computer system, what is called a difference file (difference program) may be sufficient.

  1, 1a: Cell measurement system, 10: Display unit, 20, 20a: Imaging flow cytometer, 21, 21a, 21b, 21c, 21d: Flow path, 22, 22a: Imaging unit, 23: Image acquisition unit, 24: Storage unit, 25: three-dimensional image generation unit, 26: light source, 27, 27a: imaging element, 28, 28a: light modulation unit, 29: signal acquisition unit, 30: image generation unit, 200, 200a: control unit, CL ... cells, L1, L2, L3 ... optical elements, OG ... objective lenses

Claims (4)

At least one flow path through which the observation target is flown;
A light source for irradiating the channel with a band-like excitation light,
An imaging unit that images a cross section of the observation target by imaging fluorescence from the observation target that has passed through the position where the excitation light is irradiated,
An imaging flow cytometer comprising: a three-dimensional image generation unit that generates a three-dimensional image of the observation target based on a plurality of captured images of the cross section captured by the imaging unit. The imaging flow cytometer according to claim 1, wherein the observation target is sorted based on information indicating a form of the observation target indicated by the cross section captured by the imaging unit. The flow path is a plurality of flow paths arranged in parallel,
The excitation light is applied to the plurality of flow paths,
The imaging flow cytometer according to claim 1, wherein the imaging unit captures an image of the cross section of the observation target flowing through each of the plurality of flow paths. On the optical path between the light source and the imaging device that detects the intensity of the fluorescence, a light modulation unit having a plurality of regions having different light characteristics is arranged,
The imaging unit,
The image of the cross section of the observation target is reconstructed as a captured image based on the intensity of the fluorescence detected by the imaging element and the optical characteristics of the light modulation unit. An imaging flow cytometer according to any one of the preceding claims.

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